115 New MiniPromoters with Restricted Retinal Expression When Docked in the Mouse Genome Show the Same Expression When Delivered in AAV Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright ©[.]
AAV VECTORS I of AAV8-CBA-mMut at birth: 18 Mut +/- and 18 Mut -/- treated mice (>70%) subsequently developed HCCs between 12 and 21 months of life Forty-one uninjected Mut +/- mice were also followed for 18 to 25 months and only a single mouse in this group developed HCC, indicating that a strain effect could not explain the increased rate of tumorigenesis seen in the animals that received AAV8 A control group of 11 Mut +/- mice was treated in an identical manner with AAV8-CBAGFP and greater than 50% of these mice developed HCCs between 14 and 25 months The increased occurrence of HCC following the AAV mediated delivery of GFP indicates that the expression of the mMut transgene is not solely responsible for the development of HCC In addition, we have performed a pre-clinical AAV dose escalation study in mice This study revealed that increasing the AAV8 dose from 1x109-10 GC/pup (7x1011-12 GC/kg) [n=16] to 1-2x1011 GC/pup (approx 1x1014 GC/kg)[n=19] caused a corresponding increase in the occurrence of HCC from 12% to 84%, respectively While numerous toxicology studies in a variety of species have demonstrated the safety of AAV vectors, a small number of publications have documented hepatic tumorigenesis in mice treated with AAV, and one report has implicated insertional mutagenesis by an AAV vector as a factor in the development of HCC in a small number of mice Although the relative doses of AAV used in our pre-clinical studies are higher than those being administered in current clinical trials, it is likely that the treatment of many genetic diseases via systemic delivery of AAV will require higher doses than the doses that are currently being given in clinical trials to achieve a therapeutic effect We are in the process of identifying the sites of vector integration in the HCCs to assess the distribution of AAV integration and whether there may be a possibly relationship to carcinogenesis Determining why AAV gene delivery in mice is infrequently associated with increased tumorigenesis and if such toxicity is relevant to humans will lead to improved safety in AAV gene delivery 114 A Novel Adeno-Associated Virus Serotype (AAV)-8 Capsid Mutant Improves Human Coagulation Factor IX Expression In Vivo Dwaipayan Sen,1 Rupali A Gadkari,2 Govindarajan Sudha,2 Nishanth Gabriel,1 V Ramya,3 Sukesh C Nair,3 N Srinivasan,2 Alok Srivastava,1,4 Giridhara R Jayandharan.1,4 Hematology, Christian Medical College, Vellore, India; Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, India; 3Immunohematology and Transfusion Medicine, Christian Medical College, Vellore, India; 4Center for Stem Cell Research, Christian Medical College, Vellore, India Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/ proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid scAAV8EGFP vectors containing the wild-type (WT) and each one of the S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of X 1010 vgs/ animal in C57BL/6 mice in vivo The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B) The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor , Kupffer cells and TLR-9] In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B Figure 1: Efficacy of the novel K137R-AAV8 vector in comparison to the WT-AAV8 vector 115 New MiniPromoters with Restricted-Retinal Expression When Docked in the Mouse Genome Show the Same Expression When Delivered in AAV Elizabeth M Simpson,1,2,3 Charles N de Leeuw,1,2 Frank M Dyka,4 Sanford L Boye,4 Stephanie Laprise,1 Michelle Zhou,1 Alice Y Chou,1 Lisa Borretta,1 Simone C McInerny,1 Elodie PortalesCasamar,1 Magdalena I Swanson,1 Steven J M Jones,2,5,6 Robert A Holt,2,3,5,6 Daniel Goldowitz,1,2 William W Hauswirth,4 Wyeth W Wasserman.1,2 Centre for Molecular Medicine and Therapeutics at the Child and Family Research Institute, University of British Columbia, Vancouver, BC, Canada; 2Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada; Department of Psychiatry, University of British Columbia, Vancouver, BC, Canada; 4Department of Ophthalmology, College of Medicine, University of Florida, Gainesville, FL; 5Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, BC, Canada; 6Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada Small human promoters that drive cell-type restricted gene expression will be critical for the success of many clinical gene therapies This report is focused on the development of such MiniPromoters for retinal research and gene therapy We have tested the hypothesis that the human Pleiades MiniPromoters selected for expression in the brain, would be a rich source of new promoters for the retina We also tested the hypothesis that brain-restricted S47 AAV VECTORS I promoters such as the Pleiades Promoters, which were developed using single-copy site-specific knock-in to the mouse genome, would exhibit the same expression pattern when used in adeno-associated virus (AAV) We provide data in support of both hypotheses Initially, MiniPromoters were designed using computational biology, generated by fusion PCR from human BACs, cloned to drive reporters EGFP and/or lacZ, and targeted 5’ of Hprt in mouse embryonic stem cells from which mice were derived Expression analysis was performed in male mice at embryonic day 12.5, and adult brain and eye Viral expression of MiniPromoters was tested in recombinant AAV2 driving hGFP and packaged in AAV2(Quad Y-F) (Petrs-Silva et al., Mol Ther., 2011) The viruses were injected intravitreally into four-week old C57BL/6J mice We have developed 18 new MiniPromoters capable of driving expression in the brain when docked single-copy site-specifically in the mouse genome In addition, we have further characterized the expression pattern of 15 previously published MiniPromoters (Portales-Casamar et al., PNAS, 2010) Taken together, we have identified 17 new MiniPromoters with eye expression Of these, three with restricted expression in the ganglioncell layer were tested driving expression from the AAV genome and found to maintain their restricted expression We conclude that the Pleiades MiniPromoters, which were initially selected for brain expression, represent a rich and unique resource of promoters able to express in the retina We further conclude that either the computational method of MiniPromoter design, and/or characterization of those promoters using single-copy site-specific integration in the mouse genome, renders them unusually suitable for use in AAV and likely other viruses Finally, the MiniPromoters are entirely human-DNA sequence and so should be particularly useful for clinical applications 116 AAV Serotypes 1, 8, and Share a Conserved Mechanism for Axonal Transport but Exhibit Differences in Distal Gene Transfer In Vivo Michael J Castle,1,2 April Giles,1,2 Erika L F Holzbaur,1 John H Wolfe.1,2 University of Pennsylvania, Philadelphia, PA; 2Children’s Hospital of Philadelphia, Philadelphia, PA The axonal transport of AAV vectors to distal brain regions following intraparenchymal injection is frequently observed but poorly understood Some serotypes, such as AAV9, are known to be transported along specific axonal pathways, but other serotypes, such as AAV8, often fail to transport along these same pathways and exhibit limited distal transduction Due to a lack of understanding of this variability, it is difficult to predict where gene transfer will occur in the brain prior to injection, particularly with strongly-transported serotypes such as AAV9 In order to investigate this process in vivo, we examined neurons of the entorhinal cortex (EC) which extend axons into the dentate gyrus of the hippocampus (DG) Adult C57BL/6 mice were unilaterally injected with 1.8x1010 GC of either AAV8 or AAV9 into either the DG or the EC, and AAV genomes were detected by in situ hybridization Both serotypes strongly transduced cells at the site of injection Following injection of AAV9 into the DG, distal transduction was observed in the EC, indicating that AAV9 was endocytosed at axon termini in the DG and transported retrograde to cell bodies in the EC Furthermore, following injection of AAV9 into the EC, distal transduction was observed in the DG, indicating that AAV9 was transported anterograde and released from axon termini in the DG, where it transduced second-order cells In contrast, AAV8 exhibited minimal retrograde transport and no anterograde transport, highlighting the clear differences that exist between serotypes To further investigate the cellular mechanisms underlying these differences, we used a microfluidic cell culture system to examine axonal transport following specific application of dye-labeled AAVs to cell bodies or axon termini of rat E18 cortical neurons Serotypes 1, 8, and were observed to undergo S48 fast axonal transport in both directions The character of transport was conserved among these serotypes, with similar velocities, run lengths, and pauses Furthermore, pairs of green and red dye-labeled AAVs of serotypes 1, 8, and were coinfected in mass cultures of rat E18 cortical neurons, and the extent to which each pair of serotypes co-transported along the axon was quantified A high degree of colocalization was observed during axonal transport in both directions for all pairs of serotypes, providing further evidence that these AAVs utilize the same compartments and motors for axonal transport As all three serotypes can undergo axonal transport once endocytosed, this suggests that differences in uptake at the plasma membrane underlie the differences in distal transduction observed in vivo Thus, it may be possible to enhance distal gene transfer simply by increasing the amount of AAV that is injected locally, and conversely, to restrict transduction to the injected area by decreasing the amount of injected AAV The non-reciprocal axons which project from the EC to the DG offer an ideal system for the ongoing study of this process in vivo 117 AAV6 Captron Vectors Facilitate Persistent Expression of Therapeutic Levels of Human α1Antitrypsin in Canine Lungs Christine L Halbert,1 David K Madtes,2 A D Miller.1 Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA Lung-targeted gene therapy for human alpha-1 antitrypsin (hAAT) deficiency has been modeled in dogs exposed to an AAV6 vector encoding hAAT Dog lung epithelial cells are highly susceptible to AAV6 vector transduction, and bronchial delivery of the AAV6 vector encoding hAAT resulted in therapeutic levels of hAAT in the epithelial lining fluid of dog lungs Transient immune suppression dramatically increased AAV vector-mediated levels and duration of hAAT An AAV capsid-specific CTL response was detected after, but not during, the immune suppression, which correlated with a drop in hAAT to subtherapeutic levels, suggesting that cellular immunity to AAV capsid limited long term hAAT expression Here we tested whether AAV6 vectors made using a capsid gene with a large intron (captron gene), which eliminates contaminating capsid-expressing genes might further improve the levels and duration of hAAT expression in canine lungs Indeed, two dogs that received an AAV6 vector encoding hAAT that was produced using the captron gene continued to express therapeutic levels of hAAT in their lung fluid months to year after immune suppression was stopped, and no CTL response was observed Analysis of lung tissue showed persistence of hAAT in lung alveolar cells These results support the hypothesis that captron vectors are beneficial in lowering the immunogenicity of AAV vectors in vivo Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy ... developed 18 new MiniPromoters capable of driving expression in the brain when docked single-copy site-specifically in the mouse genome In addition, we have further characterized the expression. .. tested driving expression from the AAV genome and found to maintain their restricted expression We conclude that the Pleiades MiniPromoters, which were initially selected for brain expression, ... axon termini in the DG and transported retrograde to cell bodies in the EC Furthermore, following injection of AAV9 into the EC, distal transduction was observed in the DG, indicating that AAV9 was